Efficient protein depletion by genetically controlled deprotection of a dormant N‐degron

Taxis, Christof; Stier, Günter; Spadaccini, Roberta; Knop, Michael

doi:10.1038/msb.2009.25

Cited by 93 publications

(111 citation statements)

References 47 publications

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“…We analyzed how the Ras/cAMP/PKA pathway affects meiotic cell divisions and spore formation. For this purpose, we employed a recently developed method that allows the meiosis-specific depletion of proteins (27,59). Specifically, we created strains that either destabilized both Ras1 and Ras2 (referred to as Ras) during meiosis or contained only a single Tpk subunit, Tpk2, which was depleted accordingly (referred to as Tpk).…”

Section: Resultsmentioning

confidence: 99%

“…Here, we used a method which allows the control of Ras/cAMP/PKA pathway activity during meiosis by the destabilization of key components after meiotic entry. The destabilization of target proteins is achieved by the exposure of a dormant Ndegron by the site-directed proteolysis of a degradation tag using the tobacco etch virus (TEV) protease (59). The meiosis-specific destabilization of target proteins relies on the usage of the IME2 promoter for the expression of the TEV protease (27).…”

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confidence: 99%

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Acetate Regulation of Spore Formation Is under the Control of the Ras/Cyclic AMP/Protein Kinase A Pathway and Carbon Dioxide in Saccharomyces cerevisiae

2012

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In Saccharomyces cerevisiae, the Ras/cyclic AMP (cAMP)/protein kinase A (PKA) pathway is a nutrient-sensitive signaling cascade that regulates vegetative growth, carbohydrate metabolism, and entry into meiosis. How this pathway controls later steps of meiotic development is largely unknown. Here, we have analyzed the role of the Ras/cAMP/PKA pathway in spore formation by the meiosis-specific manipulation of Ras and PKA or by the disturbance of cAMP production. We found that the regulation of spore formation by acetate takes place after commitment to meiosis and depends on PKA and appropriate A kinase activation by Ras/Cyr1 adenylyl cyclase but not by activation through the Gpa2/Gpr1 branch. We further discovered that spore formation is regulated by carbon dioxide/bicarbonate, and an analysis of mutants defective in acetate transport (ady2⌬) or carbonic anhydrase (nce103⌬) provided evidence that these metabolites are involved in connecting the nutritional state of the meiotic cell to spore number control. Finally, we observed that the potential PKA target Ady1 is required for the proper localization of the meiotic plaque proteins Mpc70 and Spo74 at spindle pole bodies and for the ability of these proteins to initiate spore formation. Overall, our investigation suggests that the Ras/cAMP/PKA pathway plays a crucial role in the regulation of spore formation by acetate and indicates that the control of meiotic development by this signaling cascade takes places at several steps and is more complex than previously anticipated.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Acetate Regulation of Spore Formation Is under the Control of the Ras/Cyclic AMP/Protein Kinase A Pathway and Carbon Dioxide in Saccharomyces cerevisiae

2012

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“…To assess the contribution of PD-mediated WUS movement to SAM activity, we developed a system to interfere with WUS protein function in a cell type-specific manner based on the TIPI-Degron approach (31,32). Previously, WUS mobility was modulated either by increasing protein size or hampering mobility by the addition of a nuclear localization signal (NLS) (19).…”

Section: Wus Activity In Stem Cells Is Required For Meristem Maintenamentioning

confidence: 99%

A mechanistic framework for noncell autonomous stem cell induction in Arabidopsis

Daum

Medzihradszky

Suzaki

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

294

261

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Cell-cell communication is essential for multicellular development and, consequently, evolution has brought about an array of distinct mechanisms serving this purpose. Consistently, induction and maintenance of stem cell fate by noncell autonomous signals is a feature shared by many organisms and may depend on secreted factors, direct cell-cell contact, matrix interactions, or a combination of these mechanisms. Although many basic cellular processes are well conserved between animals and plants, cell-to-cell signaling is one function where substantial diversity has arisen between the two kingdoms of life. One of the most striking differences is the presence of cytoplasmic bridges, called plasmodesmata, which facilitate the exchange of molecules between neighboring plant cells and provide a unique route for cell-cell communication in the plant lineage. Here, we provide evidence that the stem cell inducing transcription factor WUSCHEL (WUS), expressed in the niche, moves to the stem cells via plasmodesmata in a highly regulated fashion and that this movement is required for WUS function and, thus, stem cell activity in Arabidopsis thaliana. We show that cell context-independent mobility is encoded in the WUS protein sequence and mediated by multiple domains. Finally, we demonstrate that parts of the protein that restrict movement are required for WUS homodimerization, suggesting that formation of WUS dimers might contribute to the regulation of apical stem cell activity.transcription factor mobility | cell-to-cell trafficking | shoot apical meristem | SAM

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“…Platform for an ORFeome library protein inactivation methods were developed, such as 'TEV protease-induced protein inactivation' (TIPI) (Taxis et al, 2009) and 'degrade Green Fluorescent Protein' (deGradFP) (Caussinus et al, 2011). These methods require the attachment of a degron unit to the target protein or the creation of a GFP (or close derivative) fusion with the target protein, respectively.…”

Section: Research Articlementioning

confidence: 99%

A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila

et al. 2013

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SUMMARYOverexpression screens are used to explore gene functions in Drosophila, but this strategy suffers from the lack of comprehensive and systematic fly strain collections and efficient methods for generating such collections. Here, we present a strategy that could be used efficiently to generate large numbers of transgenic Drosophila strains, and a collection of 1149 UAS-ORF fly lines that were created with the site-specific ΦC31 integrase method. For this collection, we used a set of 655 genes that were cloned as two variants, either as an open reading frame (ORF) with a native stop codon or with a C-terminal 3xHA tag. To streamline the procedure for transgenic fly generation, we demonstrate the utility of injecting pools of plasmids into embryos, each plasmid containing a randomised sequence (barcode) that serves as a unique identifier for plasmids and, subsequently, fly strains. We also developed a swapping technique that facilitates the rapid exchange of promoters and epitope tags in vivo, expanding the versatility of the ORF collection. The work described here serves as the basis of a systematic library of Gal4/UAS-regulated transgenes.

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Efficient protein depletion by genetically controlled deprotection of a dormant N‐degron

Cited by 93 publications

References 47 publications

Acetate Regulation of Spore Formation Is under the Control of the Ras/Cyclic AMP/Protein Kinase A Pathway and Carbon Dioxide in Saccharomyces cerevisiae

Acetate Regulation of Spore Formation Is under the Control of the Ras/Cyclic AMP/Protein Kinase A Pathway and Carbon Dioxide in Saccharomyces cerevisiae

A mechanistic framework for noncell autonomous stem cell induction in Arabidopsis

A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila

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